Perhaps this is another dumb question, but I may need help to see the difference between Sc.3, Sc.11,Sc.16 and Vma.3,Vma.11,Vma.16.
Are they different names to identify the same proteins?
Probably it’s explained somewhere in the paper but maybe I missed it. Hopefully I’m slowly learning to read the biology research paper format, but I’m not there yet. Your explanations have been helpful, though. Thanks.
More dumb questions:
Does the experiment show that Anc3-11 is interchangeable with the combination of Vma3 and Vma11 ?
If that’s the case, where is the functional complexity increase?
Isn’t the system functioning either way about the same?
Also, if i use 5 screws of type A in one case,
Or 4 screws A and one screw B in another.
But in both cases I get the same results, that’s fine. I’ve done that when repairing old stuff while lacking the exact parts required in the documentation. As long as I can get the thing to work, without having to figure out how to build it from scratch, I’m a happy camper. The functionality remains. I don’t see the point quite well yet.
Maybe I’m missing something again. It’s challenging to understand biology research papers while lacking a solid background in biology.
I really appreciate that you’re answering my questions so clearly in this conversation
I agree the nomenclature can be a bit confusing. Apparently they are using Vma.X to refer to non-species-specific protein homologues, and Sc.X to refer specifically to the versions of the those proteins found in the yeast species Saccharomyces cerevisiae.
Yes. It can perform the function of both.
In the modern version of the V-ATPase found in fungi, it requires both Vma3 and Vma11 proteins present. If either one of them is missing, the system stops working. So they both require the presence of the other. So in modern V-ATPases in fungi, three protein coding genes are necessary to perform the function of the hexameric ring complex: Vma16, Vma11, and Vma3.
The phylogeny of the proteins implied that the ancestral version of the V-ATPase only required two protein coding genes, Anc16 and Anc.3-11.
The reconstructed ancestral protein Anc.3-11 can functionally replace either of Vma3 and Vma11, and even perform the function of both of them combined:
When the resurrected Anc.3–11 was transformed into yeast deficient for Vma3 ( vma3 Δ) or Vma11( vma11 Δ), growth in the presence of elevated CaCl2 was rescued, indicating that the functions of the present-day Vma3 and Vma11 proteins were already present before the duplication that generated them (Fig. 2a). Furthermore, Anc.3–11—unlike either of its present-day descendants—can partially rescue growth in yeast that are doubly deficient for both Vma3 and Vma11 ( vma3 Δ vma11 Δ).
If that’s the case, where is the functional complexity increase? Isn’t the system functioning either way about the same?
The V-ATPase used to require two protein coding genes to encode the function of the hexameric ring complex. It now requries three protein coding genes to encode that function in fungi.
Also, if i use 5 screws of type A in one case,
Or 4 screws A and one screw B in another.
But in both cases I get the same results, that’s fine. I’ve done that when repairing old stuff while lacking the exact parts required in the documentation. As long as I can get the thing to work, without having to figure out how to build it from scratch, I’m a happy camper. The functionality remains. I don’t see the point quite well yet.
Using a screw analogy, imagine you have to screw in six screws to secure some construction. The screw holes are two different sizes, so you use two different size screws. Then you’re handed a new version of that construction with three different size holes, so now you need three different size screws instead of two. You still need 6 screws in total, but three different sizes in stead of two. That’s the sense in which the complexity of the construction increased. It still performs the same function, it just requires more different types of screws.
2 Likes
Your answer clarified it for me. Thanks.
I got confused unnecessarily, because after reading your answer I noticed they had made that distinction too, but perhaps the penny didn’t drop in my mind until I read your explanation. Thanks.
Yes, I see your point. Thanks.
I[quote=“Rumraket, post:25, topic:4011”]
I agree the nomenclature can be a bit confusing. Apparently they are using Vma.X to refer to non-species-specific protein homologues, and Sc.X to refer specifically to the versions of the those proteins found in the yeast species Saccharomyces cerevisiae .
[/quote]
Is there any difference in AA sequence between Sc3 Sc11 Sc16 and their corresponding Vma3 Vma11 Vma16?
Thanks.
Tried the uniprot to no avail.
They’re accessed in GenBank. Make sure to set it to All proteins, instead of nucleotides.
You can find the species-specific protein names used in supplementary materials section S1. The whole list.
Here’s a protein sequence from the first item on the list, M_musculus_16 NP_291095:
ORIGIN
1 mtglellylg ifvafwacmv vvgicytifd lgfrfdvawf ltetspfmws nlgiglaisl
61 svvgaawgiy itgssiiggg vkapriktkn lvsiifceav aiygiimaiv isnmaepfsa
121 tepkaighrn yhagysmfga gltvglsnlf cgvcvgivgs gaaladaqnp slfvkilive
181 ifgsaiglfg vivailqtsr vkmgd
//
That’d be one of the Vma.16 proteins. We can compare that to the Sc.16 protein from yeast, S_cerevisiae_16 NP_011891:
ORIGIN
1 mnkeskdddm slgkfsfshf lyylvlivvi vyglyklftg hgsdinfgkf llrtspymwa
61 nlgialcvgl svvgaawgif itgssmigag vraprittkn lisiifcevv aiygliiaiv
121 fsskltvata enmysksnly tgyslfwagi tvgasnlicg iavgitgata aisdaadsal
181 fvkilvieif gsilgllgli vgllmagkas efq
//
Alignment says they’re ~47% identical.
2 Likes
Excellent! Thanks!!!
I see each line shows up to 60 ?
You’re teaching a fast online course on proteins here. 
I’m learning quite a lot from this conversation.
Hopefully other readers can use this learning opportunity too.
1 Like
Thanks, my pleasure. I’ve learned a lot from online discussions myself.
2 Likes
Are all these comparisons within the fungi group?
Less than half of the AA sequence of Sc.16 matches Vma.16?
Does this difference remain relatively consistent for all the other comparisons between different fungi organisms?
Does that tell us anything about the required AA positions for the given functions?
What about the uncolored AA positions? Are they useless or we don’t know?
Now that you have provided so detailed instructions on how to use the available tools, I’ll try to find time to analyze this carefully, but probably it will take me long to get through it all without rush.
Again thanks for sharing all that helpful technical information here.
As you stated before, this is fascinating.
And for me it’s quite a learning experience.
BTW, I wonder why there aren’t more “like” checks in your posts. Aren’t there more interested readers here?
1 Like
No, a substantial portion of these sequences are from animals. If you look at S2 in supplementary materials you can see the phylogeny of all protein sequences used, and from what clades the species belong to.
Less than half of the AA sequence of Sc.16 matches Vma.16?
For some arbitrarily picked eukaryote outside of the fungi clade, yes. Generally speaking, more closely related fungi are more likely to have more similar proteins of the same type of course.
You could probably find more close relatives of Saccharomyces cerevisiae with more similar Vma16 subunits.
Does this difference remain relatively consistent for all the other comparisons between different fungi?
No. Within fungi, you can easily find that the same subunits will be more similar. Though fungi still have some very deep branches separating different fungal clades. And these fungi have unicellular lifestyles and grow and divide very fast, easily several generations per day, so a lot of molecular evolution can happen between them compared to two arbitrarily picked mammals in the same amount of time.
To pick an example, the fungal species Aspergillus fumigatus subunit 16 protein, and the Aspergillus terreus subunit 16 proteins are ~88% identical (both being molds). But between Aspergillus fumigatus, and Saccharomyces cerevisiae (which is a yeast), they are about 60% identical.
Does that tell us anything about the required AA positions for the given functions?
It does give indications. A multiple sequence alignment for all the proteins would indicate areas under more and less constraint. Though you can’t really say that any single amino acid is absolutely required because they all depend on which other amino acids are present, and all possible combinations are practically impossible to test.
2 Likes
They’re almost definitely not useless. They are under less constraint, which implies that change to these positions are less likely to disturb the function of the protein in any significant degree. But that doesn’t mean they can be completely dispensed with. It is difficult to make really definitive statements about them without having to do colossal screening of mutants.
Now that you have provided so detailed instructions on how to use the available tools, I’ll try to find time to analyze this carefully, but probably it will take me long to get through it all without rush.
Again thanks for sharing all that helpful technical information here.
As you stated before, this is fascinating.
And for me it’s quite a learning experience.
BTW, I wonder why there aren’t more “like” checks in your posts. Aren’t there more interested readers here?
Haha, thank you for the kind words but don’t worry about it.
2 Likes
I’m sure there are many people following along.
1 Like
I don’t know enough about the subject for a “like”. But I am following the discussion.
2 Likes
I liked the graphical illustration you added, which makes it easier for non biologists to get the point about what kind of complexity the given paper is addressing: structural components, not functional complexity. Did I understand this correctly?
Was there or could there have been an ancestor ATPase that used Anc3-11, Anc16, Anc…?
Is the modern ATPase (that uses Sc3 Sc11 Sc16) functionally more complex than its ancestor (which used Anc3-11 Anc…).
Regarding the ATPase, are Sc3 Sc11 Sc16 more functionally complex than Anc3 Anc11 Anc3-11 Anc16 as far as it’s currently known?
At what point in evolutionary history did the ATPase functionality described in the given paper appear on the scene?
What preceded it? What was their functional difference? Was some important function added, removed, modified? How could we explain such a functional change?
Thanks.
I don’t know enough about the subject for anything besides asking questions* out of curiosity.
(*) perhaps dumb?
I’m following the conversation too.
But if Mr. Rumraket’s clear explanations help somebody to understand this topic better, wouldn’t it be nice to acknowledge that with a simple click on the “like” icon?
But if Mr. Rumraket’s clear explanations help somebody to understand this topic better, wouldn’t it be nice to acknowledge that with a simple click on the “like” icon at the bottom of his insightful posts?
Or is it that nobody else except me is leaning from his detailed explanations? If that’s the case then my ignorance of this topic is much worse than the average, 
That is correct. The basic function of the V-ATPase complex has remained the same over the time period in which the studied changes took place, but the number of different structural components required to perform that function has increased.
Was there or could there have been an ancestor ATPase that used Anc3-11, Anc16, Anc…?
The ancestral version of the structure used Anc3-11 and Anc16 only. If you look at the phylogeny you can also see that an even older (though not reconstructed and tested) version of the structure only used one type of protein for the hexameric ring, which we could name Anc3-11-16, the last common ancestor of all three proteins. That means at some point it only used six copies of the same protein (only one “screw” type). So over the time, the structural complexity has increased from one, to two, to three different proteins required.
Is the modern ATPase (that uses Sc3 Sc11 Sc16) functionally more complex than its ancestor (which used Anc3-11 Anc…).
It doesn’t look like it. The function it performs seems to have remained essentially unchanged. It consumes ATP (splits ATP into ADP+phosphate) to pump protons (H+ ions) across lipid membranes wherever it is found.
It should be noted though that ATPases are actually also ATP-synthetases, which means they can “run in reverse” and actually make ATP (combine ADP with phosphate) by being powered by the proton current. I’m not sure whether the ATPases studied here have lost that ability.
Over even longer timescales than those explored in the discussed study, the ATPase/ATP-synthetase structure has in fact changed in functional complexity by independently acquiring or losing biochemical and structural functions in different lineages. To pick just one example, in many bacteria the ATPase structure works as a sort of “engine” that powers a flagellum. The ATPase/ATP-synthetase structure is ubiquitous in cellular life.
Regarding the ATPase, are Sc3 Sc11 Sc16 more functionally complex than Anc3 Anc11 Anc3-11 Anc16 as far as it’s currently known?
I suppose that depends on how you would measure the functional complexity of an individual protein. The protein seems to be doing the same essential job as it’s ancestor, by constituting a structural component in the hexameric ring. Some alterations to how this job is performed have happened as it has mutated over time, but essentially it’s role is the same: It constitutes a structural element in the hexameric ring.
At what point in evolutionary history did the ATPase functionality described in the given paper appear on the scene?
Some time before the last universal common ancestor of all known cellular life on Earth. It isn’t known how far back it actually goes, only that it goes back before even the common ancestor that unites all cellular life. It’s older than 3.5 billion years.
As far as I am aware, ATPsynthase/ATP-synthetase proteins are the only other universally conserved proteins across the tree of life besides core translation system components like the ribosome, translation-initiation factors, tRNA, and tRNA-synthetases.
What preceded it? What was their functional difference
Was some important function added, removed, modified? How could we explain such a functional change?
Admittedly this is still very speculative. But a structural analysis of the components that make up ATPase/ATP-synthetase structures reveals homologous relationships to RNA helicases (the alpha-beta subunit hexamer responsible for ATP synthesis or cleavage exhibits the same 3dimensional structure as enzymes that “unwind” RNA double strands) and membrane embedded protein/RNA translocases(the hexameric ring discussed above exhibits a 3dimensional structure very similar to proteins that transport proteins or other molecules in and out of cells). This would imply the ultimate origin of ATPase/ATP-synthetase is essentially the combination of two such independent structures. This hypothesis has not been tested though.
You can read about that here:
Mulkidjanian AY, Makarova KS, Galperin MY, Koonin EV. Inventing the dynamo
machine: the evolution of the F-type and V-type ATPases. Nat Rev Microbiol. 2007
Nov;5(11):892-9. Review. DOI: 10.1038/nrmicro1767
Accessed freely here.
3 Likes
Do you know of papers explaining how such a change could have occurred? Thanks.
Can’t they test it like they did it in the discussed paper? It would be fascinating too.